We have recently shown that calcium and calmodulin stimulate the endogenous phosphorylation of acetylcholine receptor enriched membranes prepared from Torpedo, Californica. We believe this is a novel finding which must be exploited to assess its impact on understanding the function of the neuromuscular junction. In particular we propose to study the relationship of this phenomenon to acetylcholine receptor structure and function. We have found that the major protein species that are phosphorylated in the presence of calcium and calmodulin correspond to the subunits of the acetylcholine receptor of Torpedo, Californica by SDS polyacrylamide gel electrophoresis. We will now use several different techniques to further identify the phosphorylated species including the use of specific antibodies directed against the acetylcholine receptor. Furthermore, we will identify the protein kinase, the protein phosphatase and the calmodulin binding components present in our membranes as well as the specific sites of phosphorylation by protein fingerprinting techniques. These findings will be related to acetylcholine receptor structure. We will attempt to purify the kinase and phosphatase and reconstitute these activities in artificial membranes along with purified receptor and other natural substrates. A membrane associated calmodulin plus calcium regulated kinase has never been purified and this would represent an opportunity to study the properties of a novel enzymatic species. Of special interest will be the role of phospholipid in the activity of this kinase. Further we will examine several hypotheses concerning the role of calcium plus calmodulin stimulated phosphorylation in acetylcholine receptor function. One hypothesis is that phosphorylation of receptor affects the rate constants for the allosteric interconversions of the acetylcholine receptor. Another hypothesis is that the phosphorylation of the receptor is involved in the structure or localization of the receptor or its interaction with the cytoskeleton. We will also further our studies on the direct inhibitory effect of the phenothiazines on the acetylcholine receptor channel.